Acute lung injury and its more severe form, acute respiratory distress syndrome (ARDS), are devastating illnesses with high rates of incidence and mortality. Patients with acute lung injury are typically provided supplemental oxygen using positive pressure mechanical ventilation, but this can lead to additional injury, termed ventilator- induced lung injury (VILI). The long term objective of this proposal is to improve understanding of the mechanisms by which the combination of exposure to high levels of oxygen (hyperoxia) and overdistention (or stretch) of lung cells contributes to ventilator-induced lung injury. The central hypothesis of this application is that hyperoxia induces structural changes in alveolar epithelial and endothelial cells, as well as macrophages, that alter their mechanical properties making them more susceptible to injury caused by mechanical stretch. Mechanisms of the initiation of cell injury will be investigated using primary cultures of mouse alveolar type II (AT2) epithelial cells, primary human lung endothelial cells, mouse alveolar and bone marrow-derived macrophages, cultures of mouse lung slices, and a mouse model of combined hyperoxia and VILI. In Aim 1 we will test the hypothesis that exposure of cells or lung slices causes changes in cell structural elements that increase the elastic modulus of the cells through activation of RhoA. We will measure the Young?s modulus, an indication of an object?s ability to deform, using atomic force microscopy in the indentation mode, and we will determine how hyperoxia changes cytoskeletal structures including f-actin, microtubules, and focal adhesions. In Aim 2 we will investigate how hyperoxia increases stretch-induced cell detachment and injury. In Aim 3 we will test the hypothesis that RhoA-mediated changes in structure and mechanical properties increases lung injury in mice in a combined model of hyperoxia and VILI. The proposed studies will investigate the biophysical mechanisms that contribute to lung injury during mechanical ventilation and provide new insights into mechanotransduction, the process of converting mechanical signals to biological signals.